Neuronal TNFR1 promotes neuroinflammation and the onset of EAE
With the aim of interpreting the opposing neurotoxic and neuroprotective effects of the TNF cytokine system described in the literature, we studied the effects of neuronal TNFR1 and TNFR2 using two CNS demyelination models, experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination (CPZ). We crossed TNFR1ff and TNFR2ff mice with CamkII-Cre mice to achieve TNFR1 or TNFR2 depletion selectively in glutamatergic excitatory CNS neurons. Neuron-specific TNFR1KO (nTNFR1KO) and TNFR2KO (nTNFR2KO) mice were born at the expected Mendelian ratio, were viable, fertile and didn’t display any spontaneous phenotypes. Efficiency and specificity of TNFR1 and TNFR2 deletion in brain and spinal cord tissues was verified by DNA analysis which showed recombination specifically in brain and spinal tissues, and not any other tissue tested (Supplementary Figure 1).
nTNFR1KO, nTNFR2KO, and their respective TNFR1ff and TNFR2ff littermate controls were immunized with MOG to induce EAE, a disease driven by myelin-reactive T cells and other infiltrating immune effector cells. In our MOG-EAE model in B6 mice pathology is characterized by severe infiltration of the meninges and spinal cord parenchyma by immune cells (including CD4+ and CD8+ T cells, B cells, NK1.1+ cells and myeloid cells), microglia and astrocyte activation, demyelination and axonal damage in the white matter at the peak of disease. Pathology partially resolves during the chronic phase of disease [30].
nTNFR1KO, nTNFR2KO and control mice all showed full susceptibility to EAE (Figure 1). nTNFR1KO mice showed a small but significant delay in disease onset compared to TNFR1ff controls in 2 of 3 experiments, but thereafter the two groups of mice showed no differences in clinical disease progression up to the last time point studied (day 70) (Figure 1A). nTNFR2KO mice developed EAE with equal onset and clinical course as TNFR2ff littermate controls up to the last time point studied (day 38) (Figure 1C). The results suggest that neuronal TNFR1 plays a small disease-advancing role at the onset of EAE; otherwise, neuronal TNFR1 and TNFR2 do not have significant effects on the clinical course of EAE.
To investigate how neuronal TNFR1 advances EAE onset, RNA was isolated from the spinal cord of nTNFR1KO and TNFR1ff mice at disease peak in the control group and analyzed for the expression of early-disease marker genes [36]. nTNFR1KO and TNFR1ff spinal cord showed equally marked reduction in expression of myelin (Mbp) and neuronal (Snap25) gene markers compared to corresponding naïve mice confirming the onset of EAE (Figure 1B). However, while TNFR1ff spinal cord showed significantly increased expression of the inflammatory gene marker (H2-Ab1) at disease peak compared to naïve, nTNFR1KO showed no increase (Figure 1B). These results suggest that neuronal TNFR1 promotes an acute neuroinflammatory response in spinal cord tissue involving microglia and astrocytes, and thereby the onset of clinical symptoms in EAE, while neuronal TNFR2 has no obvious effect on disease development.
Neuronal TNFR1 advances the onset of microglia responses and demyelination, and is necessary for OLG loss and axon damage in cuprizone demyelination
We next used nTNFR1KO, nTNFR2KO, and control mice in an acute toxicity demyelination model induced by 6-weeks of dietary CPZ [32]. CPZ is a copper chelator that causes mitochondrial dysfunction and oxidative stress, both of which are key features of progressive MS [42]. In B6 mice CPZ induces acute responses of microglia and astrocytes, massive death of mature OLG between weeks 2-5 of CPZ feeding that is thought to be triggered by immune mechanisms [43], and predictable primary demyelination in the midline corpus callosum, followed by almost complete remyelination by 2 weeks after removal of CPZ from the diet [11, 32].
CPZ-induced pathology in the corpus callosum of nTNFR1KO mice was overall less severe than in TNFR1ff controls. nTNFR1KO mice showed less demyelination at CPZ3 by LFB staining of myelin (Figure 2Ai&ii), a time point where myelin detected by the more sensitive CNPase immunostaining method appeared still well preserved in both strains. nTNFR1KO mice showed less OLG and myelin loss at CPZ5 by CNPase immunostaining, compared to TNFR1ff controls (Figure 2Bi&ii). nTNFR1KO mice also showed a reduced microglia response at CPZ3 by Iba1 immunostaining (Figure 2Ci&ii), and no measurable axon damage at any time point by numbers of axonal APP spheroids, compared to controls (Figure 2Di&ii). Both strains showed full resolution of pathology by 2 weeks after cessation of CPZ feeding (CPZ6+2) indicating that remyelination proceeds normally. These results indicate that the combination of solTNF/TNFR1 signaling and CPZ induces stress in neurons that results in the exacerbation of CPZ-induced pathology.
Considering that both TNF signaling and oxidative stress are strong inducers of autophagy [44, 45], and that autophagy in microglia is responsible for the degradation and clearance of myelin debris in vitro [46], we also investigated the effect of neuronal TNFR1 on autophagy in CPZ demyelination. We measured autophagy levels in the brain sections from naïve and CPZ-fed nTNFR1KO and control mice by immunostaining for LC3, a key component of autophagosomes. Autophagic induction was significantly up-regulated in cells located in the corpus callosum (glial cells) of CPZ5 control mice compared to naïve CPZ0 controls, measured by the numbers of LC3-immunoreactive puncta per LC3-positive cell (Figure 3). On the other hand, levels of autophagic induction were similarly low in CPZ5 and naïve CPZ0 nTNFR1KO mice, as in naïve control mice, showing an absence of up-regulation. These results suggest that neuronal TNFR1 is necessary for stimulating autophagy in glial cells, probably in myelin-phagocytosing microglia and reflecting the increased levels of demyelination and need for clearance of myelin debris in the control animals.
nTNFR2KO and TNFR2ff mice showed no differences in CPZ-induced pathology, measured by LFB staining, or CNPase, Iba1, and APP immunostaining (Supplementary Figure 2A-D) and both strains showed full resolution of pathology by 2 weeks after cessation of CPZ feeding (CPZ6+2).
These results show that as in EAE, neuronal TNFR1 promotes neuroinflammation and demyelination in the CPZ model. Notably, neuronal TNFR1 contributes to acute microglia responses and is required for damage of both axons and OLG in this model. The effect of neuronal TNFR1 on axon damage might be direct by increasing the oxidative stress initiated by CPZ in neurons themselves, and indirect in both neurons and OLG by increasing microglia responses and the production of pro-inflammatory cytokines and chemokines. As in EAE, neuronal TNFR2 has no effect on disease development in the CPZ demyelination model.
Neuronal IKKβadvances neuroinflammation and the onset of demyelination in cuprizone demyelination
The pro-inflammatory effects of TNFR1 are dominantly mediated through activation of the transcription factor NF-κB, which in turn induces expression of genes encoding cytokines, chemokines and anti-apoptosis molecules [22]. Under disease conditions it is induced and is a critical mediator of inflammation. To examine the link between neuronal TNFR1 and NF-κB signaling in CNS demyelinating disease, we crossed mice carrying a conditional floxed allele for IKKβ (IKKβff mice), the main NF-κB activating kinase in the canonical NF-κB pathway [22] with CamkII-Cre mice, to generate nIKKβKO mice [30]. We previously showed that nIKKβKO mice have a small but significant delay in EAE onset similar to that described here for nTNFR1KO mice, but unlike nTNFR1KO mice nIKKβKO mice subsequently develop a severe non-remitting disease, indicative of neuroprotective effects of neuronal IKKβ during chronic disease [30].
Here we induced CPZ demyelination in nIKKβKO and IKKβff mice. At the onset of pathology at CPZ3, nIKKβKO mice showed reduced demyelination by LFB staining of myelin (Figure 4Ai&ii), axon damage by numbers of APP-immunoreactive spheroids in axons (Figure 4Di&ii), and neuroinflammation by Iba1 immunostaining of microglia (Figure 5Ai&ii) and GFAP immunostaining of astrocytes (Figure 5Di&ii), compared to IKKβff controls, effects that resemble those in nTNFR1KO mice. Unlike nTNFR1KO mice, nIKKβKO mice subsequently developed full pathology with loss of myelin and OLG measured at CPZ5 by CNPase immunostaining (Figure 4B). This was further shown by immunostaining for apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase that contributes to apoptosis and has been implicated in CPZ-induced OLG death [43]. AIF-immunoreactivity was increased in the corpus callosum of both IKKβff and nIKKβKO mice at CPZ5, most probably in oligodendrocytes which are cells that undergo apoptosis in this model (Figure 4Ci&ii).
Analysis of whole brain RNA levels for disease marker genes, first for myelin, showed that naïve (CPZ0) nIKKβKO mice express higher levels of Mbp in brain compared to IKKβff mice (Supplementary Figure 3A). Expression of Mbp and Olig2 dropped during CPZ feeding and increased again in both nIKKβKO and IKKβff mice after CPZ removal, showing myelin recovery independently of neuronal IKKβ (Supplementary Figure 3A, B). Expression of the neuron-specific gene Snap25, which encodes a protein essential for synaptic function, sharply dropped during CPZ feeding and increased again in both groups after CPZ removal. Interestingly, the rebound increase of Snap25 expression in nIKKβKO mice was consistently much higher than in controls (Figure 4E), suggesting that neuronal NF-κB might inhibit recovery of neuronal functions following CPZ toxicity. Expression of inflammatory markers Tnf, Ccl2 and Cxcl16 in control IKKβff mice showed increases at two distinct time points, first at CPZ2 at the initiation of microglia responses (Tnf,Ccl2, Cxcl16), and again at CPZ5 at the peak of demyelination/initiation of remyelination (Ccl2, Cxcl16), or CPZ6+0,5 during remyelination (Tnf) (Figure 5B, C, E). Neither Tnf nor Ccl2 showed initial increase in expression at CPZ2 in nIKKβKO mice, correlating with the delay in glial cell activation observed in these mice at CPZ3 (Figure 5B, C). Also, levels of Ccl2 and Cxcl16 were significantly lower in nIKKβKO mice at the peak of disease at CPZ5 (Figure 5C, E). These results support the pathological findings that neuronal IKKβ promotes neuroinflammation induced by CPZ feeding.
T cells, and specifically IL-17-producing T cells, are reported to participate in CPZ demyelination [47], so we investigated T cell infiltration by counting numbers of CD3-immunoreactive cells in the corpus callosum. Infiltrating CD3-immunoreactive T cells were localized in the corpus callosum of control IKKβff mice at CPZ5, and infiltration was resolved after CPZ withdrawal at CPZ6+1 and CPZ6+4 (Supplementary Figure 3Ci&ii). Infiltrating CD3-immunoreactive T cells were also localized in the corpus callosum of nIKKβKO mice, but unlike in control IKKβff mice, continued to accumulate after CPZ withdrawal at CPZ6+1, showing late resolution at CPZ6+4 (Supplementary Figure 3Ci&ii).
These data show that neuronal NF-κB activity, like neuronal TNFR1, plays a role in initiating neuroinflammation (microglia and astrocytes), demyelination and axon damage in response to dietary CPZ, and might have additional, different roles to TNFR1 during disease resolution and recovery.
Neuronal TNFR2 increases preconditioning protection against seizures and the survival of hippocampal neurons in kainic acid-induced excitotoxicity
Evidence for neuroprotective properties of TNF stems mainly from in vitro studies in which solTNF pretreatment of enriched neuron cultures (TNF preconditioning) protects them against a wide range of metabolic, excitotoxic and oxidative death stimuli and promotes maintenance of calcium homeostasis [14, 15]. To investigate the role of neuronal TNFR1 or TNFR2 in glutamate excitotoxicity in vivo, we first used an acute kainic acid (KA) excitotoxicity model. KA is a non-degradable glutamate analogue that induces epileptic seizures and death of CA3/CA2 hippocampal neurons in susceptible mouse strains. B6 mice are known to exhibit high seizure scores but no excitotoxic neuron death [34]. Also, low doses of KA are known to protect mice against seizures induced by subsequent higher doses of KA [33], allowing us also to examine the effect of neuronal TNFR in preconditioning protection against glutamate excitotoxicity in vivo.
Acute systemic (i.p.) administration of KA (20 or 24 mg/kg) consistently induced seizures in groups of nTNFR1KO, nTNFR2KO and control mice without high mortality. No differences in seizure activity between nTNFR1KO (Figure 6A, grey circles) and TNFR1ff controls (Figure 6A, black circles) or nTNFR2KO (Figure 5B, grey circles) and TNFR2ff controls (Figure 6B, black circles) were observed in this acute model during the 90 minutes of monitoring, although a trend for less severe scores was noted in nTNFR2KO mice compared to TNFR2ff controls (Figure 6B).
We next used a KA preconditioning protocol, adapted from one previously described [33]. In TNFR2ff controls low dosage KA (15 mg/kg, i.p.) significantly reduced severity of seizures induced by subsequent high dosage KA (20 mg/kg, i.p.) (Figure 6B, black squares) compared to high dosage KA alone (Figure 6B, black circles), an effect consistent with KA preconditioning protection. In nTNFR2KO mice however, the preconditioning protection effect of low dosage KA on seizures induced by subsequent high dosage KA (Figure 6B, grey squares) compared to high dosage KA alone (Figure 6B, grey circles) was reduced and non-significant. The loss of KA preconditioning protection in nTNFR2KO mice appeared to be a combined effect of reduced acute seizure intensity and reduced preconditioning protection, but these results provide the first indication that neuronal TNFR2 is necessary for effective preconditioning neuroprotection induced by low-dose KA during glutamate excitotoxicity in vivo. A direct protective role for neuronal TNFR2 was further supported by the presence of frequent pyknotic neurons stained by cresyl violet (Nissl), a feature of neuron death, localized in patches in both CA2 and CA3 regions of the hippocampus of nTNFR2KO, but not TNFR2ff mice, 5 days after KA seizures with and without preconditioning (Figure 6C).
Astrocyte TNFR1 and tmTNF via neuronal TNFR2 are necessary for, and a TNFR2 agonist reproduces, TNF preconditioning protection of cortical neurons against NMDA excitotoxicity in vitro.
To reconcile previously reported neuroprotective effects of solTNF (and therefore TNFR1) against glutamate excitotoxicity in vitro [14, 15], with the in vivo findings of TNFR2 neuroprotection here, we modeled different cellular TNF/TNFR interactions in vitro using astrocyte-neuron co-cultures and measured glutamate excitotoxicity induced by NMDA in neurons. Mouse cortical neurons and astrocytes were isolated from WT mice, or mice deficient in TNF (TNFKO), soluble TNF (tmTNFKI), TNFR1 (TNFR1KO) or TNFR2 (TNFR2KO), and different combinations of these cells were used for co-cultures.
In this system, exposure of WT astrocyte-neuron co-cultures to NMDA (50 μM) supplemented with glycine (10 μM) for 24 h induced neuron death, measured by increased percentage of NeuN-positive neurons with pyknotic nuclei stained by Hoechst (Supplementary Figure 4). Astrocyte viability was not affected by NMDA (data not shown), and neuron death was strongly inhibited by the non-competitive NMDA antagonist MK801 (Supplementary Figure 4). Consistent with previous studies [10, 48], pretreatment of WT B6 neuron-astrocyte co-cultures with soluble recombinant hTNF or mTNF (100 ng/ml) for 24 h prior to NMDA challenge, consistently and equally protected neurons by 17-20% against NMDA death, and hTNF was used for all experiments described below (Supplementary Figure 4; Figure 6).
Neuroprotection by hTNF was absent when WT neurons were cultured with TNFKO astrocytes but maintained in WT neurons cultured with tmTNFKI astrocytes, and in TNFKO, tmTNFKI, as well as TNFR1KO neurons cultured with WT astrocytes (Figure 7A). Importantly, neuroprotection was reduced when TNFR2KO neurons were cultured with WT astrocytes (Figure 7A). These results suggest that preconditioning protection of cortical neurons induced by exogenous soluble hTNF is mainly dependent on astrocyte tmTNF-neuronal TNFR2 interactions. Under these conditions, neuronal TNFR1 also has a very small neuroprotective effect, detectable in WT astrocyte- TNFR2KO neuron co-cultures (Figure 7A).
To understand how soluble hTNF triggers this mechanism, we performed experiments using TNFR1KO neurons and astrocytes. hTNF signals efficiently through mouse TNFR1 but not through TNFR2, due to species specificity of mouse TNFR2 [49]. Absence of TNFR1 from astrocytes, not neurons, abolished protection (Figure 7B). Together the results suggest that soluble hTNF engages astrocyte TNFR1, inducing activation and production of tmTNF. solTNF has been previously shown to increase the expression of tmTNF in cell lines [50]. Astrocyte tmTNF-neuronal TNFR2 interactions then precondition neurons for protection against glutamate excitotoxicity (Diagram 1).
To confirm the neuroprotective role of neuronal TNFR2, we compared pretreatment of WT astrocyte-neuron co-cultures at NA-DIV7 with a novel TNFR2 agonist (TNFR2ag 100 ng/ml) [51], or with soluble hTNF (100 ng/ml), for 24 h prior to NMDA challenge. As previously described [40, 52], TNFR2ag pretreatment induced strong neuroprotection against NMDA death, equal to that induced by soluble hTNF (Figure 7C). Together these results suggest that astrocyte tmTNF-neuronal TNFR2 interactions dominantly mediate preconditioning protection of neurons against glutamate excitotoxicity in vitro, and likely in vivo. Neuronal TNFR1 has limited neuroprotective effect in models of glutamate excitotoxicity.